Rapid plant rna extraction kit

Total RNAs were extracted from roots, stems, leaves, petals, ovules and SAM, using a rapid plant RNA extraction kit (Aidlab, Beijing, China). The cDNAs were synthesized from total RNA using a first-strand cDNA synthesis kit (TaKaRa, Dalian, China), and then subjected to real-time PCR analyses. Real-time PCRs were performed on a CFX96 real-time PCR detection system using SYBR Green Supermix (Bio-Rad, CA, USA) according to the manufacturer’s introductions. The thermal cycling parameters were: 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s, 57 °C for 20 s, followed by a standard melting curve to monitor PCR specificity. The primers are listed in Additional file 2: Data S1. Data were analyzed using the software Bio-Rad CFX Manager 2.0 provided by the manufacturer.

Upland cotton RNAs were extracted from roots, stems, leaves, petals, ovules, and fibers of different developmental stages using a rapid plant RNA extraction kit (Aidlab, Beijing, China). The cDNAs were synthesized from total RNA using a first-strand cDNA synthesis kit (TaKaRa, Dalian, China), and then subjected to real-time PCR analyses. Real-time PCRs were performed on a CFX96 real-time PCR detection system using SYBR Green Supermix (Bio-Rad, CA, USA) according to the manufacturer’s introductions. The thermocycling parameters were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s, 57°C for 20 s, followed by a standard melting curve to monitor PCR specificity. Cotton histone3 (AF024716) [42 (link)] and GhUBQ14 [43 (link)] genes were amplified as internal standards. Reactions were performed for three replicates. Data were analyzed using the software Bio-Rad CFX Manager 2.0 provided by the manufacturer.

Total RNA was extracted from 0.2 g seeds, seed coats, embryos, and buds using a rapid plant RNA extraction kit (Aidlab, Beijing, China). The single-stranded cDNAs were synthesized from 1 μg of RNA using a cDNA synthesis kit according to the manufacturer's instructions (Takara Biotechnology, Dalian, China). qRT-PCR was performed with a gene specific primer pair and a β-actin primer pair as an internal control (Table 1). Reactions were performed on a CFX96 real-time PCR detection system with SYBR Premix Ex Taq (Takara). The thermo cycling parameters were as follows: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C for denaturation and 1 min at 60°C for annealing and extension. The specificity of the PCR was assessed by the presence of a single peak in the dissociation curve after the amplification and by size estimation of the amplified product. The comparative cycle threshold (CT) method (2-ΔΔCT) method was used to quantify cDNAs with amplification efficiencies equivalent to that of the reference actin gene. Each experiment was repeated at least three times using the same cDNA source. For every reaction, the mean and SE values of relative transcript abundance were calculated. All the primers were designed by Beacon Designer 7 software. Results presented are the average of three independent biological replicates repeated three times.

Total RNAs were extracted from various cotton tissues using a rapid plant RNA extraction kit (Aidlab, Beijing, China). First-strand cDNAs were generated from 1 μg total RNA using a PrimeScript™ RT reagent kit (TaKaRa, Dalian, China) with gDNA eraser to degrade trace genomic DNAs according to the instructions of the manufacturer. Quantitative PCRs were performed with SYBR-Green PCR Mastermix (Vazyme, Nanjing, China). Amplification was monitored on a real-time basis using a CFX96 real-time PCR system (Bio-Rad, California, United States). The thermocycling parameters were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 5 s, 57°C for 20 s, and a standard melting curve program was added to monitor PCR specificity. GhACT was used as an internal control (Artico et al., 2010 (link)). The qRT-PCR results were analyzed using Bio-Rad CFX Manager 2.0 provided by the manufacturer (Bio-Rad, California, United States). The primers are listed in Supplementary Table 1.

Total RNAs were extracted from various cotton tissues using a rapid plant RNA extraction kit (Aidlab, Beijing, China), and genomic DNA degradation and first‐strand cDNA synthesis were performed using a PrimeScript™ RT reagent kit with gDNA eraser (TaKaRa, Dalian, China). Quantitative PCRs were performed in a CFX96 real‐time PCR system (Bio‐Rad, CA) using SYBR Green Supermix. The thermocycling parameters were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s, 57 °C for 20 s and a standard melting curve to monitor PCR specificity. GhACT4 and GhUBQ14 were used as reference to normalize the transcript levels of target genes (Artico et al., 2010). The PCR results were analysed using Bio‐Rad CFX Manager 2.0 provided by the manufacturer (Bio‐Rad). Primers used for quantitative PCRs are listed in Table S6.